DISTINCT SUBPOPULATIONS OF THE RETINOBLASTOMA PROTEIN SHOW A DISTINCTPATTERN OF PHOSPHORYLATION

Phosphorylation of the retinoblastoma protein (pRB) is assumed to regu late its growth-controlling function. Moreover, hypophosphorylated and hyperphosphorylated forms of pRB can be distinguished by virtue of th e distinct affinities with which they bind to the cell nucleus. This p roperty allows the identification of individual cell nuclei that conta in pRB in one or the other form. We show here that after cells emerge from a quiescent (G(0)) state, conversion of their complement of pRB i nto a hyperphosphorylated form occurs in late G(1), preceding entry in to S phase by several hours. Thus, contrary to earlier reports, pRB ph osphorylation is not co-ordinated with the G(1)-S transition and may n ot directly regulate it. A distinct set of phosphopeptides is found ex clusively in those forms of pRB that show the loose nuclear associatio n characteristic of the hyperphosphorylated form of pRB. Another set o f phosphopeptides is found with both hypophosphorylated and hyperphosp horylated forms. This suggests the existence of distinct patterns of p hosphorylation that are associated with different subsets of pRB molec ules. We conclude that substantial phosphorylation of pRB exists in G( 1) even prior to the hyperphosphorylation point. Cyclin-dependent kina ses can cause a liberation of pRB from cell nuclei in vitro. Phosphory lation by members of this kinase family is therefore likely to be dire ctly involved in the change in nuclear affinity in vivo and the associ ated changes in pRB functioning.